Method for preventing and treating diabetes using neurturin

ABSTRACT

The present invention relates generally to methods for preventing and/or treating pancreatic disorders, particularly those related to diabetes, by administering a neurturin protein product.

The present invention relates generally to methods for preventing and/ortreating pancreatic disorders, particularly those related to diabetes,by administering a neurturin product.

BACKGROUND OF THE INVENTION

The pancreas is an exocrine gland that secretes digestive enzymesdirectly into the digestive tract as well as an endocrine gland thatsecretes hormones into the blood stream. The exocrine function isassured by acinar and centroacinar cells that secrete various digestiveenzymes via intercalated ducts into the duodenum. The functional unit ofthe endocrine pancreas is the islet of Langerhans. Islets are scatteredthroughout the exocrine portion of the pancreas and are composed of fourmain cell types: alpha-, beta-, delta- and PP-cells (reviewed forexample in Kim & Hebrok, 2001, Genes Dev. 15: 111-127). Beta-cellsproduce insulin, represent the majority of the endocrine cells and formthe core of the islets, while alpha-cells secrete glucagon and arelocated in the periphery. Delta-cells and PP-cells are less numerous andsecrete somatostatin and pancreatic polypeptide, respectively. Recently,cells producing the neuropeptide Ghrelln have been found in pancreaticislets (Wierup et al., 2002, Regal Pept. 107:63-9.).

Early pancreatic development has been well studied in different species,including chicken, zebrafish, and mice (for a detailed review, see Kim &Hebrok, 2001, supra). The pancreas develops from distinct dorsal andventral anlagen. Pancreas development requires specification of thepancreas anlage along both anterior-posterior and dorsal-ventral axes. Anumber of transcription factors, that are critical for proper pancreaticdevelopment have so been identified (see Kim & Hebrok, 2001, supra;Wilson et al., 2003, Mech Dev, 120: 65-80).

In humans, the acinar and ductal cells retain a significantproliferative capacity that can ensure cell renewal and growth, whereasthe islet cells become mostly mitotically inactive. This is in contrastto rodents where beta-cell replication is an important mechanism in thegeneration of new beta cells. It has been suggested, that duringembryonic development, pancreatic islets of Langerhans originate fromdifferentiating duct cells or other cells with epithelial morphology(Bonner-Weir & Sharma, 2002, J Pathol. 197: 519-526; Cu et al., 2003,Mach Day. 120: 35-43). In adult humans, new beta-cells arise in thevicinity of ducts (Butler at al., 2003, Diabetes 52: 102-110; Bouwens &Pipeleers 1998, Diabetologia 41: 629-633). However, also an intra-isletlocation or an origin in the bone marrow has been suggested forprecursor cells of adult beta-cells (Zulewski et al., 2001, Diabetes 50:521-533; lanus at al., 2003, J Clin Invest. 111: 843-850). Pancreaticislet growth is dynamic and responds to changes in insulin demand, forexample during pregnancy or due to changing body weight duringchildhood. In adults, there is a good correlation between body mass andislet mass (Yoon et al., 2003, J Clin Endocrinol Metab. 88: 2300-2308).

Pancreatic beta-cells secrete insulin in response to blood glucoselevels. Insulin amongst other hormones plays a key role in theregulation of the fuel metabolism. Insulin leads to the storage ofglycogen and triglycerides and to the synthesis of proteins. The entryof glucose into muscles and adipose cells is stimulated by insulin. Inpatients who suffer from diabetes mellitus type I or LADA (latentautoimmune diabetes in adults (Pozzilli & Di Mario, 2001, Diabetes Care.8:1460-67) beta-cells are being destroyed due to autoimmune attack. Theamount of insulin produced by the remaining pancreatic islet cells istoo low, resulting in elevated blood glucose levels (hyperglycemia). Indiabetes type II liver and muscle cells loose their ability to respondto normal blood insulin levels (insulin resistance). High blood glucoselevels (and also high blood lipid levels) in turn lead to an impairmentof beta-cell function and to an increase in beta-cell apoptosis. It isinteresting to note that the rate of beta-cell neogenesis does notappear to change in type II diabetics (Butler et al., 2003 supra), thuscausing a reduction in total beta-cell mass over time. Eventually theapplication of exogenous insulin becomes necessary in type II diabetics.

Improving metabolic parameters such as blood sugar and blood lipidlevels (e.g. through dietary changes, exercise, medication orcombinations thereof) before beta-cell mass has fallen below a criticalthreshold leads to a relatively rapid restoration of beta-cell function.However, after such a treatment the pancreatic endocrine function wouldremain impaired due to the only slightly increased regeneration rate.

In type I diabetics, where beta-cells are being destroyed by autoimmuneattack, treatments have been devised which modulate the immune systemand may be able to stop or strongly reduce islet destruction (Raz etal., 2001, Lancet 3513: 1749-1753; Chatenoud et al., 2003, Nat RevImmunol. 3: 123-132; Homann et al., Immunity. 2002, 3:403-15). However,due to the relatively slow regeneration of human beta-cells suchtreatments can only be successful if they are combined with agents thatcan stimulate beta-cell regeneration.

Diabetes is a very disabling disease, because today's commonanti-diabetic drugs do not control blood sugar levels well enough tocompletely prevent the occurrence of high and low blood sugar levels.Out of range blood sugar levels are toxic and cause long-termcomplications like for example renopathy, retinopathy, neuropathy andperipheral vascular disease. There is also a host of related conditions,such as obesity, hypertension, heart disease and hyperlipidemia, forwhich persons with diabetes are substantially at risk.

Apart from the impaired quality of life for the patients, the treatmentof diabetes and its long term complications presents an enormousfinancial burden to our healthcare systems with rising tendency. Thus,for the treatment of, type I and type II diabetes as well as for latentautoimmune diabetes in adults (LADA) there is a strong need in the artto identify factors that induce regeneration of pancreatic insulinproducing beta-cells. These factors could restore normal function of theendocrine pancreas once its function is impaired or event could preventthe development or progression of diabetes type I, diabetes type II, orLADA.

In this invention, we disclose a novel and so far unknown use for theneurotrophic factor neurturin to stimulate the formation or regenerationof insulin producing beta-cells and thus, a use in the treatment andprevention of diabetes.

Neurotrophic factors are growth factors that regulate the survival andmaintain the phenotypic differentiation of certain nerve and/or glialcell populations (Varon at al., 1978, Ann. Rev. Neuroscience 1: 327-361;Thoenen at al., Science, 229:238-242, 1985). Nerve growth factor (NGF)was the first neurotrophic factor to be identified and characterized(Levi-Montalcini at al., 1951, J. Exp. Zool. 116:321). The second memberof this family to be discovered was brain-derived neurotrophic factor(Leibrock at al., 1989, Nature 341:149-152).

Glial-derived neurotrophic factor (GDNF)—a neurotrophic factorstructurally unrelated to NGF—was discovered during a search for factorscrucial to the survival of midbrain dopaminergic neurons, whichdegenerate in Parkinson's disease (Lin at al., 1993, Science260:1130-2). Sequence analysis revealed it to be a distant member of thesuperfamily of transforming growth factor 8 (TGF-beta) factors.

Another neurotrophic factor that is structurally closely related to GDNFand unrelated to NGF is neurturin (Kotzbauer et al., 1996, Nature 384:467-470). Neurturin, GDNF and two other related factors (artemin andpersephin) define a family of neurotrophic factors referred to asTGF-beta-related neurotrophins. These neurotrophic factors promote thesurvival of various neurons including peripheral autonomic and sensoryneurons as well as central motor and dopamine neurons, and have beenproposed as therapeutic agents for neurodegenerative diseases (seereview by Takahashi, 2001, Cytokine Growth Factor Rev 12(4):361-73; seealso, for example, U.S. Pat. No. 6,090,778 and EP1005358B1, thedisclosures of which are hereby incorporated by reference).TGF-beta-related neurotrophins signal through a unique two-receptorcomplex consisting of a glycosylphosphatidylinositol-linked cell surfacemolecule, the GDNF family receptor alpha (GFRalpha) and receptor proteintyrosine kinase Ret.

Apart from the described functions in neuronal tissue GDNF/RETsignalling is crucial for the differentiation of certain non-neuronaltissues. For example, GFRalpha1 and the Ret are both necessary receptorcomponents for ureteric bud outgrowth and subsequent branching in thedeveloping kidney (Catalano at al., 1998, Neuron 21:53-62; Tang at al.,1998, J Cell Biol. 142 (5):1337-45). Ret, GFRalpha-1 (the GDNFreceptor), and GFRalpha-2 (the Neurturin receptor) are expressed bytesticular germ cells, while GDNF and Neurturin are expressed by Sertolicells. Both GDNF and Neurturin stimulate DNA synthesis in spermatogonia.Furthermore, GFRalpha, GFRalpha ligands and co-receptors are expressedin germ cell tumors and thus may act as paracrine factors inspermatogenesis (Viglietto at al., 2000, Int J. Oncol. 16(4):689-94).

Only recently, it was shown that the biology of GDNF signalling is muchmore complex than originally assumed. GDNF family ligands also signalthrough the neural cell adhesion molecule NCAM. In cells lacking Ret,GDNF binds with high affinity to the NCAM and GFRalpha1 complex (seereview by Sariola & Saarma, 2003, J Cell Sol. 116(Pt 19):3855-62).Signalling via the c-met receptor kinases has also been demonstrated(see Popsueva et al., 2003, J Cell Biol. 161(1):119-29).

Although it has been discussed in the prior art that GDNF/RET signallingis crucial for the differentiation of neuronal and certain non-neuronaltissues, it has not been disclosed that a member of family ofTGF-beta-related neurotrophins is involved in the regeneration ofpancreatic tissue. We found surprisingly, that neurturin stimulates theformation or regeneration of insulin producing pancreatic beta-cellswhich play an essential role in diabetes. Thus, in this invention, wedisclose the use of neurturin in the treatment and prevention ofdiabetes.

SUMMARY OF THE INVENTION

The present invention relates to new methods for stimulating and/orinducing the differentiation of progenitor cells, e.g. stem cells intoinsulin-producing cells or for promoting the protection, survival and/orregeneration of insulin producing cells using a neurturin product and/ora modulator/effector thereof that influences, particularly increases theexpression level or function of a neurturin protein product.

Thus, the present invention provides methods for treating patientssuffering from a disease caused by, associated with, and/or accompaniedby functionally impaired and/or reduced numbers of pancreatic isletcells, particularly insulin producing beta-cells, by administering atherapeutically effective amount of a neurturin product or a compoundthat influences the neurturin expression level or function. Functionalimpairment or loss of pancreatic islet cells may be due to e.g.autoimmune attack such as in diabetes type I or LADA, and/or due to celldegeneration such as in progressed diabetes type II. The methods of thepresent invention may also be used to treat patients at risk to developdegeneration of insulin producing beta-cells to prevent the start orprogress of such process.

The neurturin product or the effector/modulator thereof may beadministered e.g. as a pharmaceutical composition, via implantation ofneurturin product expressing cells, and/or via gene therapy.

Further, the invention relates to cell preparations comprisingneurturin-treated insulin producing cells or neurturin expressing cells.

Numerous additional aspects and advantages of the invention will becomeapparent to those skilled in the art upon consideration of the followingdescription of the Figures and detailed description of the inventionwhich describes presently preferred embodiments thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the neurturin dependent induction of the differentiation ofinsulin producing cells.

Mouse embryonic stem (ES) cells were differentiated to insulin producingcells as described previously (patent application PCT/EP02/04362,published as WO 02/086107, which is incorporated herein by reference).In differentiated cells, the abundance of insulin mRNA (FIGS. 1A and 1B)and of beta-cell glucose transporter Glut2 mRNA (FIGS. 1C and 1D) wasdetermined using quantitative RT-PCR in two independent examples. Levelswere normalized using 18S RNA as control and a cycle number of 36 asreference. The numbers on the vertical line refer to the abundance ofthe indicated transcripts relative to an abundance for which 36 cyclesare necessary for detection, ‘wt ES’ refers to unmodified mouse R1embryonic stem (ES) cells; ‘Pax4 ES’ refers to R1 mouse embryonic stem(ES) cells stably transfected with a CMV-Pax4 expression construct;‘insulin expression rel. to delta Ct36’ refers to expression of insulin;‘Glut2 expression rel. to delta Ct36’ refers to expression of beta-cellglucose transporter; ‘ES’ refers to mouse embryonic stem cells, asdescribed in Example 1; ‘control’ refers to the differentiation protocolas described in Example 2, without any addition of neurturin; ‘EB+NTN’refers to the differentiation protocol as described in Example 3, withthe addition of neurturin to embryoid bodies).

FIG. 2 shows the structure of the mouse mDG770 transgenic construct,Shown is the HP promoter (0.8 kb rat insulin II promoter) as a thinline, the mouse DG770 cDNA (mDG770) as white box, the hybrid intronstructure (hybrid-intron) as grey box and the polyadenylation signal(bgh-polyA) as black box.

FIG. 3 shows pancreatic islets of mDG770 transgenic mice with ectopicmDG770 expression. Taqman expression analysis on islet cDNA isolatedfrom two wild type and two transgenic littermates using a mDG770specific primer/probe pair. The data are presented as fold mDG770induction relative to wild type mDG770 expression in islets.

FIG. 4 shows random fed blood glucose levels of DG770 transgenic mice(rIP-mDG770) compared to wild type mice (wt) on high fat (HF) diet.Shown are blood glucose levels from random fed male wild type mice (♦,N=5) and rIP-mDG770 transgenic mice (▪, N=6). The data are expressed asmean blood glucose+/− standard deviation. The blood glucose levels aresignificantly lower in DG770 transgenic mice; meaning that higherexpression of DG770 in pancreatic islets of mammals will lower the bloodglucose level.

DETAILED DESCRIPTION OF THE INVENTION

Before the present invention is described, it is understood that alltechnical and scientific terms used herein have the same meanings ascommonly understood by one of ordinary skill in the art to which thisinvention belongs.

In the present invention the term “beta-cell regeneration” refers to anat least partial restoration of normal beta-cell function by increasingthe number of functional insulin secreting beta-cells and/or byrestoring normal function in functionally impaired beta-cells.

As used herein, the term “neurturin product” includes neurturin proteinproducts such as purified natural or synthetic neurturin and variantsthereof. Variants include insertion, substitution and deletion variantsand chemically modified derivatives. Variants also include recombinantproteins, for example but not limited to hybrids of neurturin and otherTGF-beta proteins (preferably from the GDNF-family). Also included areproteins or peptides substantially homologous to the human neurturinprecursor protein having the amino acid sequence published as GenBankAccession Number NP_(—)004549. The term “neurturin product” alsoincludes polynucleotides (e.g. mRNA/DNA) encoding the above describedneurturin protein product. The term “neurturin product” also includesneurturin homodimers or heterodimers of a neurturin protein product andanother protein, wherein the other protein preferably belongs to theGDNF-family.

The term “biologically active” as used herein means that the neurturinproduct induces and/or stimulates the differentiation of insulinproducing cells from progenitor, e.g. stem cells and/or promotes theprotection, survival, or regeneration of insulin producing cells, e.g.beta-cells. The biological activity of neurturin products may bedetermined as described in the Examples of the present application.

The term “substantially homologous” as used herein means having a degreeof homology to the biologically active human neurturin product resultingfrom the cleavage of the neurturin precursor having the amino acidsequence published as GenBank Accession Number NP_(—)004549 or to thehuman neuturin precursor itself, that is preferably in excess of 70%,most preferably in excess of 80%, and even more preferably in excess of90% or 95%. The degree of homology between the mouse and the humanprotein is about 91%, and it is contemplated that preferred mammalianneurturin proteins will have a similarly high degree of homology. Alsoincluded are proteins which are hybrids between neurturin and anotherTGFbeta-protein, preferably another member of the GDNF-family whichretain the stimulatory effect on islet cell formation found inNeurturin. The percentage of homology or identity between a neurturinproduct and the human neurturin protein or a precursor or a nucleic acidcoding therefor may be determined according to standard procedures, e.g.by using the BLAST algorithm. Preferably, the percentage of homology oridentity is calculated as the percentage of nucleotide or amino acidresidues found in the smaller of the two sequences so that align withidentical nucleotides or amino acid residues in the sequence beingcompared, when four gaps in a length of 100 nucleotides or amino acidsmay be introduced to assist in that alignment. Also included assubstantially homologous is any neurturin protein product which may beisolated by virtue of cross-reactivity with antibodies to the neurturinprotein product or whose genes may be isolated through hybridizationwith the gene or with segments of the gene encoding the neurturinprotein product.

In connection with the present invention, the term “progenitor cells”relates to undifferentiated cells capable of being differentiated intoinsulin producing cells. The term particularly includes stem cells, i.e.undifferentiated or immature embryonic, adult, or somatic cells that cangive rise to various specialized cell types. The term “stem cells” caninclude embryonic stem cells (ES) and primordial germ cells (EG) cellsof mammalian, e.g. human or animal origin. Isolation and culture of suchcells is well known to those skilled in the art (see, for example,Thomson et al., 1998, Science 282:1145-1147; Shamblott at al., 1998,Proc. Natl. Acad. Sci. USA 95:13726-13731; U.S. Pat. No. 6,090,622; U.S.Pat. No. 5,914,268; WO 0027995; Notarianni at al., 1990, J. Reprod.Fert. 41:51-56; Vassilieva at al., 2000, Exp. Cell. Res. 258:361-373).Adult or somatic stem cells have been identified in numerous differenttissues such as intestine, muscle, bone marrow, liver, and brain. WO03/023018 describes a novel method for isolating, culturing, anddifferentiating intestinal stem cells for therapeutic use. In thepancreas, several indications suggest that stem cells are also presentwithin the adult tissue (Gu & Sarvetnick, 1993, Development 118:33-46;Bouwens, 1998, Microsc Res Tech 43:332-336; Bonner-Weir, 2000, J. Mol.Endocr. 24:297-302).

Embryonic stem cells can be isolated from the inner cell mass ofpre-implantation embryos (ES cells) or from the primordial germ cellsfound in the genital ridges of post-implanted embryos (EG cells). Whengrown in special culture conditions such as spinner culture or hangingdrops, both ES and EG cells aggregate to form embryoid bodies (EB). EBsare composed of various cell types similar to those present duringembryogenesis. When cultured in appropriate media, EB can be used togenerate in vitro differentiated phenotypes, such as extraembryonicendoderm, hematopoietic cells, neurons, cardiomyocytes, skeletal musclecells, and vascular cells. We have previously described a method thatallows EB to efficiently differentiate into insulin-producing cells (asdescribed in patent application PCT/EP02/04362, published as WO02/086107 and by Blyszczuk at al., 2003, Proc Natl Acad Sci USA.100(3):998-1003, which are incorporated herein by reference).

The term ‘cultivation medium’ means a suitable medium capable ofsupporting growth and differentiation of stem cells. The term‘differentiation medium’ means a suitable medium for inducing thedifferentiation of stem cells into insulin-producing cells. The term‘terminal differentiation medium’ means a suitable medium for terminaldifferentiation of insulin-producing cells. Examples of preferred mediaare described in WO/023018, which are herein incorporated by reference.

In this invention, we disclose a novel and so far unknown use for theneurotrophic factor neurturin to stimulate and/or induce the formationor regeneration of insulin producing cells and thus, a use in thetreatment and prevention of diseases going along with (e.g. caused by,associated with or accompanied by) impaired beta-cell function, forexample but not limited to diabetes mellitus. More particularly, thediseases are diabetes type I, diabetes type II or LADA.

The present invention is based on the surprising finding that neurturinstimulates the differentiation of insulin producing cells from stemcells in vitro. Thus, a therapeutically effective amount of neurturinproduct may be administered to promote the regeneration of pancreaticbeta-cells or to promote the formation of insulin-producing cells fromstem cells or progenitor cells in vitro or in vivo. The presentinvention further relates to applications in the medical field thatdirectly arise from the method of the invention. Additionally, thepresent invention relates to applications for the identification andcharacterization of compounds with therapeutic medical effects ortoxicological effects that directly arise from the method of theinvention.

According to this invention the neurturin product may be administered

-   i) as a pharmaceutical composition e.g. enterally, parenterally or    topically, preferably directly to the pancreas,-   ii) via implantation of neurturin protein product expressing cells,    and/or-   iii) via gene therapy    as described in more detail below.

Further, the neurturin expression level in a patient might be influencedby a neurturin modulator/effector administered

-   i) as a pharmaceutical composition e.g. enterally, parenterally or    topically, preferably directly to the pancreas,-   ii) via cell based therapy, and/or-   iii) via gene therapy    is as described in more detail below.

The neurturin product or the neurturin modulator/effector, i.e. apharmaceutically active substance influencing, particularly increasingthe neurturin expression level or function may be administered in theabove described manner alone or in combination with anotherpharmaceutical composition useful to treat beta-cell degeneration, forexample hormones, growth factors or immune modulating agents.

A neurturin product or a modulator/effector thereof may be administeredin patients suffering from a disease going along with impaired beta-cellfunction, for example but not limited to diabetes type I, LADA, orprogressed diabetes type II. It is further contemplated that a neurturinproduct or the modulator/effector thereof may be administeredpreventively to patients at risk to develop beta-cell degeneration, likefor example but not limited to patients suffering from diabetes type IIor LADA in early stages. A variety of pharmaceutical formulations anddifferent delivery techniques are described in further detail below.

The present invention also relates to methods for differentiatingprogenitor cells into insulin-producing cells in vitro comprising

-   (a) activating one or more pancreatic genes in a progenitor, e.g.    stem cell (optional step, particularly if embryonic stem cells are    used)-   (b) aggregating said cells to form embryoid bodies (optional step,    particularly if embryonic stem cells are used)-   (c) cultivating embryoid bodies or cultivating adult stem cells    (e.g., duct cells) in specific differentiation media containing a    neurturin protein product and/or a modulator/effector thereof under    conditions wherein beta-cell differentiation is significantly    enhanced, and-   (d) identifying and selecting insulin-producing cells.

Activation of pancreatic genes may comprise transfection of a cell withpancreatic gene operatively linked to an expression control sequence,e.g. is on a suitable transfection vector, as described in WO 03/023018,which is herein incorporated by reference. Examples of preferredpancreatic genes are Pdx1, Pax4, Pax6, neurogenin 3 (ngn3), Nkx 6.1, Nkx6.2, Nkx 2.2, HB 9, BETA2/Neuro D, Isl 1, HNF1-alpha, HNF1-beta and HNF3of human or animal origin. Each gene can be used individually or incombination with at least one other gene, Pax4 is especially preferred.

Neurturin products, e.g. neurturin protein or nucleic acid products, arepreferably produced via recombinant techniques because such methods arecapable of achieving high amounts of protein at a great purity, but arenot limited to products expressed in bacterial, plant, mammalian, orinsect cell systems.

Neurturin Protein Product

Recombinant neurturin protein product forms include glycosylated andnon-glycosylated forms of the protein. In general, recombinanttechniques involve isolating the genes encoding for neurturin proteinproduct, cloning the gene in suitable vectors and/or cell types,modifying the gene if necessary to encode a desired variant, andexpressing the gene in order to produce the neurturin protein product.

Alternatively, a nucleotide sequence encoding the desired neurturinproduct may be chemically synthesized. It is contemplated that aneurturin product may be expressed using nucleotide sequences that varyin codon usage due to the degeneration of the genetic code or allelicvariations or alterations made to facilitate production of the proteinproduct by the selected cell.

Kotzbauer et al., Nature 384:467-470, describe the identification of amouse cDNA and amino acid sequence and a human cDNA and amino acidsequence for neurturin protein. The neurturin products according to thisinvention may be isolated or generated by a variety of means. Exemplarymethods for producing neurturin products useful are described in patentapplication WO 97/08196, the disclosures of which are herebyincorporated by reference. Also described are a variety of vectors, hostcells, and culture growth conditions for the expression of neuturinprotein, as well as methods to synthesize variants of neurturin proteinproduct. Additional vectors suitable for the expression of neurturinprotein product in E. coli are disclosed in Patent No. EP 0 423 980, thedisclosure of which is hereby incorporated by reference.

The molecular weight of purified neurturin indicates that in itsbiologically active form the protein is a disulfide-bonded dimer. Thematerial isolated after expression in a bacterial system is essentiallybiologically inactive, and exists as a monomer. Refolding is necessaryto produce the biologically active disulfide-bonded dimer. Processessuitable for the refolding and maturation of the neurturin expressed inbacterial systems are substantially similar to those described inWO93/06116. Standard in vitro assays for the determination of neurturinactivity are also substantially similar to those determining GDNFactivity as described in W093/06116 and in U.S. application Ser. No.08/535,681, and are hereby incorporated by reference.

Neurturin product variants are prepared by introducing appropriatenucleotide changes into the DNA encoding the polypeptide or by in vitrochemical synthesis of the desired polypeptide. It will be appreciated bythose skilled in the art that many combinations of deletions,insertions, and substitutions can be made resulting in a protein productvariant presenting neurturin biological activity.

Mutagenesis techniques for the replacement, insertion or deletion of oneor more selected amino acid residues are well known to one skilled inthe art (e.g., U.S. Pat. No. 4,518,584, the disclosure of which ishereby incorporated by reference.)

Neurturin substitution variants have at least one amino acid residue ofthe human or mouse neurturin amino acid sequence removed and a differentresidue inserted in its place. Such substitution variants includeallelic variants, which are characterized by naturally occurringnucleotide sequence changes in the species population that may or maynot result in an amino acid change.

Chemically modified derivatives of neurturin protein products also maybe prepared by one of skill in the art given the disclosures herein. Thechemical moieties most suitable for derivatization include water solublepolymers. A water soluble polymer is desirable because the protein towhich it is attached does not precipitate in an aqueous environment,such as a physiological environment. Preferably, the polymer will bepharmaceutically acceptable for the preparation of a therapeutic productor composition. One skilled in the art will be able to select thedesired polymer based on such considerations as whether thepolymer/protein conjugate will be used therapeutically, and if so, thedesired dosage, circulation time, resistance to proteolysis, and otherconsiderations. A particularly preferred water-soluble polymer for useherein is polyethylene glycol. Attachment at residues important forreceptor binding should be avoided if receptor binding is desired. Onemay specifically desire an N-terminal chemically modified protein.

The present invention contemplates use of derivatives which areprokaryote-expressed neurturin, or variants thereof, linked to at leastone polyethylene glycol molecule, as well as use of neurturin, orvariants thereof, attached to one or more polyethylene glycol moleculesvia an acyl or alkyl linkage. Pegylation may be carried out by any ofthe pegylation reactions known in the art. See, for example: Focus onGrowth Factors, 3 (2):4-10, 1992; EP 0 154 316, the disclosure of whichis hereby incorporated by reference; EP 0 401 384; and the otherpublications cited herein that relate to pegylation.

The present invention also discloses use of derivatives which areprokaryote-expressed neurturin, or variants thereof, linked to at leastone hydrophobic residue, for example fatty acid molecule, as well as useof neurturin, or variants thereof, attached to one or more hydrophobicresidues. For example, patent application published as WO 03/010185,which is hereby incorporated by reference, describes a method forproducing acylated polypeptides in transformed host cells by expressinga precursor molecule of the desired polypeptide which are then to beacylated in a subsequent in vitro step.

Polynucleotides Encoding Neurturin Protein Product

The present invention further provides polynucleotides that encodeneurturin protein products, whether recombinantly produced or naturallyoccurring.

A nucleic acid sequence encoding a neurturin protein product, canreadily be obtained in a variety of ways, including, without limitation,chemical synthesis, cDNA or genomic library screening, expressionlibrary screening, and/or PCR amplification of cDNA. These methods andothers useful for isolating such nucleic acid sequences are set forth,for example, by Sambrook et al. (Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), byAusubel et al., eds (Current Protocols in Molecular Biology, CurrentProtocols Press, 1994), and by Berger and Kimmel (Methods in Enzymology:Guide to Molecular Cloning Techniques, vol. 152, Academic Press, Inc.,San Diego, Calif., 1987). Chemical synthesis of a nucleic acid sequencewhich encodes a neurturin protein product can also be accomplished usingmethods well known in the art, such as those set forth by Engels et al.(Angew. Chem. Intl. Ed., 28:716-734, 30 1989).

Included within the scope of this invention are neurturin productpolynucleotides with the native signal sequence and other pre-prosequences as well as polynucleotides wherein the native signal sequenceis deleted and replaced with a heterologous signal sequence. Theheterologous signal sequence selected should be one that is recognizedand processed, i.e., cleaved by a signal peptidase, by the host cell.For prokaryotic host cells that do not recognize and process the nativeneurturin signal sequence, the signal sequence is substituted by aprokaryotic signal sequence selected, for example, from the group of thealkaline phosphatase, penicillinase, or heat-stable enterotoxin 11leaders. For yeast secretion, the native neurturin signal sequence maybe substituted by the yeast invertase, alpha factor, or acid phosphataseleaders. In mammalian cell expression the native signal sequence issatisfactory, although other mammalian signal sequences may be suitable.

Expression and cloning vectors generally include a nucleic acid sequencethat enables the vector to replicate in one or more selected host cells.

Neurturin Product Pharmaceutical Compositions

Neurturin product pharmaceutical compositions typically include atherapeutically effective amount of a neurturin product in admixturewith one or more pharmaceutically and physiologically acceptableformulation. In addition to the active ingredients, neurturin productpharmaceutical compositions may contain suitable pharmaceuticallyacceptable carriers comprising excipients and auxiliaries, whichfacilitate processing of the active compounds into preparations, whichcan be used pharmaceutically. Further details on techniques forformulation and administration may be found in the latest edition ofRemington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.),the disclosure of which is hereby incorporated by reference.

Once the therapeutic composition has been formulated, it may be storedin sterile vials as a solution, suspension, gel, emulsion, solid, ordehydrated or lyophilized powder. Such formulations may be stored eitherin a ready to use form or in a form, e.g., lyophilized, requiringreconstitution prior to administration. The optimal pharmaceuticalformulations will be determined by one skilled in the art depending uponconsiderations such as the route of administration and desired dosage.Such formulations may influence the physical state, stability, rate ofin vivo release, and rate of in vivo clearance of the present neurturinproteins, variants and derivatives. Other effective administrationforms, such as slow-release formulations, inhalant mists, or orallyactive formulations are also envisioned.

For example, in a sustained release formulation, the neurturin productmay be bound to or incorporated into particulate preparations ofpolymeric compounds (such as polylactic acid, polyglycolic acid, etc.)or liposomes.

Administration/Delivery Neurturin Product

The neurturin product may be administered by any suitable means,preferably enterally or parenterally or topically directly to thepancreas, as known to those skilled in the art. The specific dose may becalculated according to considerations of body weight, body surface areaor organ size. Further refinement of the calculations necessary todetermine the appropriate dosage for treatment involving each of theabove mentioned formulations is routinely made by those of ordinaryskill in the art and is within the ambit of tasks routinely performed.Appropriate dosages may be ascertained through use of the establishedassays for determining dosages utilized in conjunction with appropriatedose-response data. The final dosage regimen involved in a method fortreating the above described conditions will be determined by theattending physician, considering various factors which modify the actionof drugs, e.g., the age, condition, body weight, sex and diet of thepatient, the severity of any infection, time of administration and otherclinical factors. As studies are conducted, further information willemerge regarding the appropriate dosage levels for the treatment ofvarious diseases and conditions.

It is envisioned that the continuous administration or sustaineddelivery of a neurturin product may be advantageous for a giventreatment. While continuous administration may be accomplished via amechanical means, such as with an infusion pump, it is contemplated thatother modes of continuous or near continuous administration may bepracticed. For example, chemical derivatization or encapsulation mayresult in sustained release forms of the protein having the effect ofcontinuous presence, in predictable amounts, based on a determineddosage regimen. Thus, neurturin protein products include proteinsderivatized or otherwise formulated to effectuate such continuousadministration.

Neurturin product cell therapy, i.e. pancreatic implantation of cellsproducing neurturin protein product, is also contemplated. Thisembodiment would involve implanting cells capable of synthesizing andsecreting a biologically active form of neurturin protein product intopatients. Such neurturin protein product-producing cells may be cellsthat are natural producers of neurturin protein product or may be cellsthat are modified to express the protein. Such modified cells includerecombinant cells whose ability to produce a neurturin protein producthas been augmented by transformation with a gene encoding the desiredneurturin protein product in a vector suitable for promoting itsexpression and secretion. In order to minimize a potential immunologicalreaction in patients being administered neurturin protein product of aforeign species, it is preferred that the cells producing neurturinprotein product be of human origin and produce human neurturin proteinproduct. Likewise, it is preferred that the recombinant cells producingneurturin protein product be transformed with an expression vectorcontaining a gene encoding a human neurturin protein product. Implantedcells may be encapsulated to avoid infiltration of surrounding tissue.Human or nonhuman animal cells may be implanted in patients inbiocompatible, semipermeable polymeric enclosures or membranes thatallow release of neurturin protein product, but that prevent destructionof the cells by the patient's immune system or by other detrimentalfactors from the surrounding tissue.

Alternatively, neurturin protein product secreting cells may beintroduced into a patient in need intraportally via a percutaneoustranshepatic approach using local anaesthesia. Between 3000 and 100 000equivalent differentiated insulin-producing cells per kilogram bodyweight are preferably administered. Such surgical techniques are wellknown in the art and can be applied without any undue experimentation,see Pyzdrowski et al, 1992, New England J. Medicine 327:220-226; Heringet al., Transplantation Proc. 26:570-571, 1993; Shapiro et al., NewEngland J. Medicine 343:230-238, 2000.

In a further preferred embodiment, neurturin protein product can bedelivered directly to progenitor, e.g. stem cells in order to stimulatethe differentiation of insulin producing cells. For example, proteindelivery can be achieved by polycationic liposomes (Sells et al. (1995)Biotechniques 19:72-76), Tat-mediated protein transduction (Fawell etal. (1993) Proc. Natl. Acad. Sci. USA 91:664-668) and by fusing aprotein to the cell permeable motif derived from the PreS2-domain of thehepatitis-B virus (Oess and Hildt (2000) Gene Ther. 7150-758).Preparation, production and purification of such proteins from bacteria,yeast or eukaryotic cells are well known by persons skilled in the art.In this embodiment of the invention, neurturin may be added preferablyat concentrations between 1 ng/ml and 500 ng/ml, more preferably between10 and 100 ng/ml, e.g. at about 50 ng/ml.

Further, the invention relates to a cell preparation comprisingdifferentiated progenitor cells, e.g. stem cells exhibiting insulinproduction, particularly an insulin-producing cell line obtainable bythe method described above. The insulin-producing cells may exhibit astable or a transient expression of at least one pancreatic geneinvolved in beta-cell differentiation. The cells are preferably humancells that are derived from human stem cells. For therapeuticapplications the production of autologous human cells from adult stemcells of a patient is especially preferred. However, the insulinproducing cells may also be derived from non-autologous cells. Ifnecessary, undesired immune reactions may be avoided by encapsulation,immunosuppression and/or modulation or due to non-immunogenic propertiesof the cells.

The insulin producing cells of the invention preferably exhibitcharacteristics that closely resemble naturally occurring beta-cells.Further, the cells of the invention preferably are capable of a quickresponse to glucose. After addition of 27.7 mM glucose, the insulinproduction is enhanced by a factor of at least 2, preferably by a factorof at least 3. Further, the cells of the invention are capable ofnormalizing blood glucose levels after transplantation into mice.

The invention further encompasses functional pancreatic cells obtainableor obtained by the method according to the invention. The cells arepreferably of mammalian, e.g. human origin. Preferably, said cells arepancreatic beta-cells, e.g. mature pancreatic beta-cells or stem cellsdifferentiated into pancreatic beta-cells. Such pancreatic beta cellspreferably secrete insulin in response to glucose. Moreover, the presentinvention provides functional pancreatic cell that express glucagon inresponse to glucose. A preparation comprising the cells of the inventionmay additionally contain cells with properties of other endocrine celltypes such as alpha-cells, delta-cells and/or PP-cells. These cells arepreferably human cells.

The cell preparation of the invention is preferably a pharmaceuticalcomposition comprising the cells together with pharmacologicallyacceptable carriers, diluents and/or adjuvants. The pharmaceuticalcomposition is preferably used for the treatment or prevention ofpancreatic diseases, e.g. diabetes.

According to the present invention, the functional insulin producingcells treated with neurturin may be transplanted preferablyintrahepatic, directly into the pancreas of an individual in need, or byother methods. Alternatively, such cells may be enclosed intoimplantable capsules that can be introduced into the body of anindividual, at any location, more preferably in the vicinity of thepancreas, or the bladder, or the liver, or under the skin. Methods ofintroducing cells into individuals are well known to those of skill inthe art and include, but are not limited to, injection, intravenous orparenteral administration. Single, multiple, continuous or intermittentadministration can be effected. The cells can be introduced into any ofseveral different sites, including but not limited to the pancreas, theabdominal cavity, the kidney, the liver, the celiac artery, the portalvein or the spleen. The cells may also be deposited in the pancreas ofthe individual.

The methodology for the membrane encapsulation of living cells isfamiliar to those of ordinary skill in the art, and the preparation ofthe encapsulated cells and their implantation in patients may beaccomplished without undue experimentation. See, e.g., U.S. Pat. Nos.4,892,538, 5,011,472, and 5,106.627, each of which is specificallyincorporated herein by reference. A system for encapsulating livingcells is described in PCT Application WO 91/10425 of Aebischer et al.,specifically incorporated herein by reference. See also, PCP ApplicationWO 91/10470 of Aebischer et al., Winn et al., Exper. Neurol., 113:322-329, 1991, Aebischer et al., Exper. Neurol., 11 1:269-275, 1991;Tresco et al., ASAIO, 38:17-23, 1992, each of which is specificallyincorporated herein by reference. Techniques for formulating a varietyof other sustained- or controlled-delivery means, such as liposomecarriers, bio-erodible particles or beads and depot injections, are alsoknown to those skilled in the art.

In another embodiment gene therapy ex vivo is envisioned, i.e. thepatient's own cells may be transformed ex vivo to produce a neurturinprotein product or a protein stimulating neurturin expression and wouldbe directly reimplanted. For example, cells retrieved from the patientmay be cultured and transformed with an appropriate vector. After anoptional propagation/expansion phase, the cells can be transplanted backinto the same patient's body, particularly the pancreas, where theywould produce and release the desired neurturin protein product.Delivery by transfection and by liposome injections may be achievedusing methods, which are well known in the art. Any of the therapeuticmethods described above may be applied to any suitable subjectincluding, for example, mammals such as dogs, cats, cows, horses,rabbits, monkeys, and most preferably, humans.

Neurturin product gene therapy in vivo is also envisioned, byintroducing the gene coding for a neurturin protein product intotargeted pancreas cells via local injection of a nucleic acid constructor other appropriate delivery methods (Hefti, J. Neurobiol.,25:1418-1435, 1994). For example, a nucleic acid sequence encoding aneurturin protein product may be contained in an adeno-associated virusvector or adenovirus vector for delivery to the pancreas cells.Alternative viral vectors include, but are not limited to, retrovirus,herpes simplex virus and papilloma virus vectors. Physical transfer,either in vivo or ex vivo as appropriate, may also be achieved byliposome-mediated transfer, direct injection (naked DNA),receptor-mediated transfer (ligand-DNA complex), electroporation,calcium phosphate precipitation or microparticle bombardment (gene gun).

Immunosuppressive drugs, such as cyclosporin, can also be administeredto the patient in need to reduce the host reaction versus graft.Allografts using the cells obtained by the methods of the presentinvention are also useful because a single healthy donor could supplyenough cells to regenerate at least partial pancreas function inmultiple recipients.

The neurturin nucleic acid and protein and effectors/modulators thereofmay be administered either as a monotherapy or as a combination therapywith other pharmaceutical agents. For example, they may be administeredtogether with other pharmaceutical agents suitable for the treatment orprevention of pancreatic diseases and/or obesity and/or metabolicsyndrome, particularly with other pharmaceutical agents suitable forstimulating and/or inducing the differentiation of insulin producingcells from progenitor cells. Further, they may be administered togetherwith pharmaceutical agents which have an immunosuppressive activity,e.g. antibodies, polypeptides and/or peptidic or non-peptidic lowmolecular weight substances. Preferred examples of immunosuppressiveagents are listed in the following Table 1.

TABLE 1 Exemplary agents for immune suppression Names Mechanism2-amino-1,3-propanediol derivatives Used for preventing or treatingchronic rejection in a patient receiving an organ or tissue allo- orxenotransplant 2-amino-2[2-(4-octylphenyl)ethyl] Immunosuppression, fromaccelerated lymphocyte homing propane-1,3-diol hydrochloride40-O-(2-hydroxyethyl)-rapamycin, Sirolimus (rapamycin) derivative, usedfor acute kidney rejection; SDZ-RAD, Everolimus reduces rejection andgraft vasculopathy following heart transplantation by inhibiting cellproliferation 6-(3-dimethyl-aminopropionyl) Immunosuppressing actionuseful also for treating autoimmune forskolin disease 6-mercaptopurine(6-MP) Used to treat Crohn's disease, inflammatory bowel disease and fororgan transplant therapy A-420983 Lck-inhibitor ABX-CBL (CBL-1) Mousemonoclonal AB targeted against human T-cell, B cells, NK cells andmonocytes, for treatment of steroid-resistant graft vs host diseases,potential use in treatment of inflammatory and autoimmune disordersAlefacept (human LFA-3 IgG1 fusion Knocks out causative memoryT-lymphocytes; Used to treat psoriasis, protein) a T-cell mediatedinflammatory disorder Antisense ICAM-1 inhibitor (ISIS Mouse monoclonalAB blocks white blood cell adhesion to T-cell 2302), Enlimomab, BIRR1,surface molecule (ICAM-1r); treatment of kidney transplant rejectionAlicaforsen Antithymocyte immunoglobulin Anti-human thymocyte,immunoglobulin; used in reversal of acute (ATGAM) kidney transplantrejection and will likely be used off-label for transplant inductiontherapy Azathioprine Treatment of rheumatoid arthritis and prevention ofkidney transplant rejection, and other autoimmune or inflammatorydisorders such as inflammatory bowel disease Baohuoside-1 Flavonoid;inhibits lymphocyte activation; Ma et al., Transplantation 78: 831-838,(2004) basiliximab Monoclonal AB that binds to receptor sites onT-cells, preventing activation by transplanted tissue (renal transplant)BMS-279700 Lck-inhibitor BTI-322 Mouse derived monoclonal AB targeted toCD2 receptor; used for prevention of first-time kidney rejection, andtreatment of resistant rejection Cladribine Antimetabolite andimmunosuppressive agent that is relatively selective for lymphocytes;used to treat lymphoid malignancies, e.g., hairycell leukemia CP-690550JAK-3 inhibitor Cyclophosphamide (CTX) Immunosuppressant for treatmentof arthritis and other auto-immune disorders and cancers Cyclosporine(cyclosporin A, 11 amino acid cyclic peptide; blocks helper T-cell,cyclosporin) immunosuppressant used in organ transplant therapy andother immune diseases Daclizumab, HAT (Humanized Anti- Monoclonal ABinhibits binding of IL-2 to IL-2 receptor by binding Tac), SMARTanti-Tac, anti-CD25, to IL-2 receptor, suppresses T-cell activityagainst allografts (renal and humanized anti-IL2-receptor transplant)Dexamethasone (Decadron, Dexone, An adrenocorticoid, effectiveimmunosuppressant in various Dexasone) disorders DIAPEP-277Immunomodulatory properties Dipeptide Boronic Acid (DPBA) Proteasomeinhibitor; Wu et al., Transplantation 78: 360-366, (2004)Docosahexaenoic acid (DHA) Immunosuppressant that lowers the proportionof T cells expressing CD4 or CD8, blocks antigen recognition process;Taku et al., Journal of Agricultural and Food Chemistry 48: 1047, (2000)efalizumab T-cell modulator that target T-cells through interactionswith adhesion molecules on endothelial cell surface, target migration ofT- cells into the skin and target activation of T-cells; Used to treatPsoriasis Efomycine M Leukocyte adhesion inhibitor, Anti-inflammatoryFTY720 (oral myriocin derivative) Alters lymphocyte infiltration intografted tissues; used for prevention of organ rejection in kidneytransplants Glatiramer acetate (co-polymer-1) Synthetic peptidecopolymer; decoy that mimics structure of myelin so immune cells bindCopaxone instead of myelin; for multiple sclerosis Glial fibrillaryacidic protein (GFAP) Possesses immunosuppressive activities in diabeticanimal models; Winer et al., Nature Medicine 9: 198, (2003) Gusperimus(15-deoxyspergualin) Intravenous immunosuppressant; suppressesproduction of cytotoxic T-cells, neutrophils and macrophages HLA-B2702peptide Human peptide, blocks action of NK cells and T-cell mediatedtoxicities, used for prevention of first kidney allograft rejectionhu1124 (anti-CD11a) Humanized monoclonal antibody; targets CD11areceptor on surface of T cells to selectively inhibit immune systemrejection of transplanted organs hOKT31γ(Ala-Ala) non Fc-bindinghumanized anti CD3 antibody Infliximab Monoclonal AB, binds andinactivates human TNFalpha; used to treat Crohn's disease and rheumatoidarthritis Interferon Immunomodulatory properties ISAtx247 Used to treatautoimmune diseases such as rheumatoid arthritis and psoriasisisotretinoin Immunosuppressant, reduces ability of T cells toproliferate in response to immune challenge. Vergelli et al.,Immunopharmacology, 31: 191, (1997) L-683,742: also described as 31-Treatment of autoimmune diseases, infectious diseases and/ordesmethoxy-31-hydroxy-L-683,590 prevention of organ transplantrejections Leflunomide (ARAVA) Antiinflammatory agent Medi-500 (T10B9)Intravenous monoclonal AB that targets human T-cells; treats acutekidney rejection and graft-vs-host disease Medi-507 Intravenoushumanized AB directed against CD2 T-cell; used to treatcorticosteroidresistant graft vs host disease and prevention of kidneyrejection Methotrexate Antimetabolite used to treat Crohn's disease,severe psoriasis, and adult rheumatoid arthritis (and as an anti-cancerdrug) Mitoxantrone Antiproliferative effect on cellular immune systemincluding T-cells, B-cells and macrophages; used to treathormone-refractory prostate cancer, acute myelogenous leukemia andmultiple sclerosis mycophenolate mofetil Proliferation of T and Blymphocytes by blocking the synthesis of purine nucleotides; used inorgan transplant therapy and inflammatory bowel disease OKT4A Mousemonoclonal AB targeted against human CD4 T cell; used for prevention ofkidney transplant rejection when used in combination with otherimmunosuppressant drugs Muromonab-CD3 Monoclonal AB that binds toreceptor sites on T-cells, preventing activation by transplanted tissuePrednisolone Corticosteroid, suppresses inflammation associated withtransplant rejection Psora-4 Kv1.3-blocker Rifampicin Antibiotic; hasimmunomodulatory properties Rituximab CD20 antibody S100β possessesimmunosuppressive activities in diabetic animal models Sirolimus,Rapamycin Immunosuppressant and potent inhibitor of cytokine (e.g.IL-2)-dependent T-cell proliferation (kidney transplant)

The combination therapy may comprise coadministration of the medicamentsduring the treatment period and/or separate administration of singlemedicaments during different time intervals in the treatment period.

Administration of a neurturin protein product and/ormodulators/effectors thereof in a pharmaceutical composition to asubject in need thereof, particularly a human patient, leads to an atleast partial regeneration of pancreatic cells. Preferably, these cellsare insulin producing beta-cells that will contribute to the improvementof a diabetic state. With the administration of this composition e.g. ona short term or regular basis, an increase in beta-cell mass can beachieved. This effect upon the body reverses the condition of diabetespartially or completely. As the subject's blood glucose homeostasisimproves, the dosage administered may be reduced in strength. In atleast some cases further administration can be discontinued entirely andthe subject continues to produce a normal amount of insulin withoutfurther treatment. The subject is thereby not only treated but could becured entirely of a diabetic condition. However, even moderateimprovements in beta-cell mass can lead to a reduced requirement forexogenous insulin, improved glycemic control and a subsequent reductionin diabetic complications. In another example, the compositions of thepresent invention will also have efficacy for treatment of patients withother pancreatic diseases such as pancreatic cancer, dysplasia, orpancreatitis, if beta-cells are to be regenerated.

In a further embodiment, the present invention allows the production ofcells for the identification and/or characterisation of compounds whichstimulate beta-cell differentiation, insulin secretion and/or glucoseresponse, more particularly of compounds which increase the neurturinexpression level or function. This method is particularly suitable forin vivo testing for diagnostic applications and drug development orscreening. The compound of interest is added to suitable cells and theneurturin expression or function is determined. Alternatively, acompound of interest is added to a neurturin-treated cell and the effecton cell differentiation and/or insulin production is determined.Preferably, differentiated insulin-producing cells used. Insulin levelsin treated cells can be determined, e.g. quantified by Enzyme LinkedImmunoabsorbent Assay (ELISA) or Radio Immuno Assay (RIA). Using thismethod, a large number of compounds can be screened and compounds thatinduce neurturin expression or support the activity of neurturin leadingto a beta-cell differentiation and/or an increase in insulin secretioncan be identified readily.

In a high-throughput screening method, the cells are transfected with aDNA construct, e.g. a viral or non-viral vector containing a reportergene, e.g. the lacZ gene or the GFP gene, under regulatory control of apromoter of a gene involved in beta-cell differentiation, e.g.preferably a Pax4 promoter. The transfected cells are divided intoaliquots and each aliquot is contacted with a test substance, e.g.candidate 1, candidate 2, and candidate 3. The activity of the reportergene corresponds to the capability of the test compound to inducebeta-cell differentiation.

In a further embodiment (which may be combined with the high-throughputscreening as described above) a medium throughput validation is carriedout. Therein, the test compound is added to cells being cultivated andthe neurturin expression and/or the insulin production is determined.Following an initial high throughput assay, such as the cell based assayoutlined above where e.g. a Pax4 promoter is used as marker forbeta-cell regeneration, the activity of candidate molecules to inducebeta-cell differentiation is tested in a validation assay comprisingadding said compounds to the culture media of the embryoid bodies.Differentiation into insulin-producing cells is then evaluated, e.g. bycomparison to wild type and/or Pax4 expressing cells to assess theeffectiveness of a compound.

EXAMPLES

A better understanding of the present invention and of its manyadvantages will be had from the following examples, given by way ofillustration.

Example 1 Generation of ES Cells Expressing the Pax4 Gene

Mouse R1 ES cells (Nagy et al. (1993) Proc. Natl. Acad. Sci. USA. 90:8424-8428) were electroporated with the Pax4 gene under the control ofthe CMV promoter and the neomycin resistance gene under the control ofthe phosphoglycerate kinase 1 promoter (pGK-1).

ES cells were cultured in Dulbecco's modified Eagle's medium containing4.5 g/l glucose, 10⁻⁴ M beta-mercaptoethanol, 2 nM glutamine, 1%non-essential amino acids, 1 nM Na-pyruvate, 15% FCS and 500 U/mlleukaemia inhibitory factor (LIF). Briefly, approximately 10⁷ ES cellsresuspended in 0.8 ml phosphate buffered saline (PBS) were subjected toelectroporation with 25 μg/ml of linearized expression vector (Joyner,Gene Targeting: A Practical Approach, Oxford University Press, New York,1993). Five minutes after electroporation, ES cells were plated on petridishes containing fibroblastic feeder cells previously inactivated bytreatment with 100 μg/ml mitomycin C. One day after electroporation,culture medium was changed to medium containing 450 μg/ml G418.Resistant clones were separately isolated and cultured 14 days afterapplying the selection medium. Cells were always cultured at 37° C., 5%CO₂. These untreated and undifferentiated ES cells were used as controlthe experiment shown in FIG. 1 (referred to as ‘ES’ in FIG. 1).

Example 2 Differentiation of ES Cells into Insulin-Producing Cells(Referred to as ‘Control’ in FIG. 1)

The ES cell line R1 (wild type, ‘wt ES’ in FIG. 1) and ES cellsconstitutively expressing Pax4 (‘Pax4 ES’ in FIG. 1) were cultivated asembryoid bodies (EB) by the hanging drop method, as described in patentapplication PCT/EP02/04362, published as WO 02/086107 and by Blyszczuket al., 2003, Proc Natl Aced Sci USA. 100: 998-1003, which areincorporated herein by reference, with media as described below and inTable 2. The embryoid bodies were allowed to form in hanging dropcultures for 2 days and then transferred for three days to suspensioncultures in petri dishes. At day 5, EBs were plated separately ontogelatin-coated 6 cm cell culture dishes containing a differentiationmedium prepared with a base of Iscove modified Dulbecco's medium. Afterdissociation and replating at day 14, cells were cultured up to 40 daysin the differentiation medium prepared with a base of Dulbecco'smodified Eagle medium: Nutrient Mixture F-12 (DMEM/F12).

Example 3 Expression of Pancreas Specific Genes after Differentiation ofES Cells into Insulin-Producing Cells

Expression levels of pancreas specific genes was measured bysemi-quantitative RT-PCR analysis. Differentiated wild type ES and Pax4ES cells were collected after embryoid body formation and suspended inlysis buffer (4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7;0.5% sarcosyl, 0.1 M beta-mercaptoethanol). Total RNA was isolated bythe single step extraction method described by Chomczynski & Sacchi,1987, Anal. Biochem. 162: 156-159). mRNA was reverse transcribed usingPolyT tail primer Oligo d(T)₁₆ (PerkinElmer) and the resulting cDNA wasamplified using oligonucleotide primers complementary and identical totranscripts of beta-cell glucose transporter Glut2 and insulin. Thehouse keeping gene beta-tubulin was used as internal standard. Reversetranscription (RT) was performed with MuLV reverse transcriptase (PerkinElmer). Multiplex PCRs were carried out using AmpliTaq DNA polymerase(Perkin Elmer) as described in Wobus at al., 1997, supra. mRNA levels ofgenes encoding Glut2 and insulin were analysed using the Dynalbeads mRNADIRECT micro kit (Dynal) according to the manufacturer's instructions.

One third of each PCR reaction was separated by electrophorese. Ethidiumbromide fluorescence signals of gels were analyzed by a special software(TINA2.08e) The intensity of the ethidium bromide fluorescence signalswas determined from the area under the curve for each peak and the dataof target genes were plotted as percentage changes in relation to theexpression of the housekeeping gene beta-tubulin.

Results show that markers for beta-cell differentiation function wereexpressed at higher levels in Pax4⁺ differentiated ES cells than indifferentiated wild type ES cells demonstrating that activation of apancreatic developmental control gene renders differentiation moreefficient than for wild type ES cells (FIG. 1). Expression of Glut2 indifferentiated stem cells indicates that hormone-producing cells arecapable of responding to glucose. Expression of substantial amounts ofinsulin in differentiated stem cells indicates that differentiated cellsshow a phenotype similar to beta-cells.

Example 4 Induction of Differentiation of Insulin-Producing Cells byNeurturin (Referred to as EB+NTN in FIG. 1)

In order to study the effect of neurturin to induce beta-celldifferentiation in vitro, we have generated stable mouse embryonic stem(ES) cells expressing Pax4 under the control of the cytomegalovirus(CMV) early promoter/enhancer region as described in Example 1. Pax4 andwild type ES cells were then cultured in hanging drops or spinnercultures to allow the formation of embryoid bodies. Embryoid bodies wereformed in the presence of 50 ng/ml of neurturin solution in PBS-01% BSA.Embryoid bodies were subsequently plated, again neurturin was addedevery second day until day 12 and afterwards enzymatically dissociated,and repleted. After dissociation, cells were cultured in adifferentiation medium containing various growth factors (see Table 2for more detail). Neurturin was obtained from RDI Research DiagnosticsINC, USA, Order Number RDI-4511. Under such conditions, the expressionof insulin was significantly induced by neurturin in two independentexamples (FIGS. 1A and 1B). In addition, the addition of neurturin tothe differentiation medium did significantly enhance the expression ofthe glucose transporter Glut-2 in two independent examples (FIGS. 1C and1D). By comparison, wild type ES cells did not contain anyinsulin-producing cell at the same stage and only small numbers ofGlut-2 expressing cells. These data demonstrate that neurturin cansignificantly promote and enhance ES cells differentiation intoinsulin-producing cells compared to wild type ES cells.

The results shown in FIG. 1 clearly demonstrate a significant inductionof the differentiation of insulin-producing (FIGS. 1A and 1B) andglucose responsive (FIGS. 1C and 1D) cells, if neurturin is added toembryoid bodies. Thus, neurturin has a strong inductive effect on thedifferentiation of insulin-producing beta cells.

TABLE 2 Protocol for the induction of differentiation ofinsulin-producing cells by Neurturin. Media B1, B2 and B27 supplement,(NA-Niacinamide) are described in Rolletschek et al., 2001, Mech. Dev.105: 93-104. Stage of Coating and Day Cultivation Medium Analysis 0hanging drops Iscove + 15% RNA (200 cells/drop) FCS addition of (EScells) Neurturin (NTN) 50 ng/ml 1 2 EBs in Iscove + 15% suspension FCS +NTN, 50 ng/ml 3 4 plating of EBs Iscove + 10% gelatin FCS + NTN, coating50 ng/ml RNA (EBs) +1 medium change B1 medium + ornithine/ NTN, 50 ng/mllaminin +2 coating +3 medium change +4 +5 medium change +6 +7 mediumchange +8 dissociation B2 + B27 + RNA (1 × 6 NA + 10% FCS cm dish) +9medium change see above, withdraw FCS +10 +11 medium change +12 +13medium change +14 +15 medium change +16 +17 medium change +18 +19 mediumchange +20 +21 medium change +22 +23 medium change +24 +25 medium changeImmunofluorescence (IF); RNA +26 +27 medium change +28 IF; RNA +29medium change +30 +31 medium change +32 IF; RNA

Example 5 Functional Characterization of the DifferentiatedInsulin-Producing Cells

One important property of beta-cells is glucose responsive insulinsecretion. To test whether the Pax4 derived insulin-producing cellspossessed this glucose responsive property, an in vitro glucoseresponsive assay was performed on the differentiated cells. On the dayof the assay, the differentiation medium of 12 or 6 well plate wasremoved and the cells were washed 3 times with Krebs Ringer BicarbonateHepes Buffer (KRBH; 125 mM NaCl, 4.7 mM KCl, 1 mM CaCl₂, 1.2 mM KH₂PO₄,1.2 mM MgSO₄, 5 mM NaHCO₃, 25 mM Hepes, 0.1% BSA) supplemented with 2.8mM glucose. For pre-incubation cells were incubated in KRBH+2.8 mMglucose for 2 hours at 37° C. Afterwards cells were incubated in 750 μlup to 1 ml KRBH+2.8 mM glucose for 1 hour at 37° C. The supernatant waskept for measurement of basal insulin secretion. For the stimulatedinsulin release 750 μl up to 1 ml KRBH containing 16.7 mM glucose wasadded to the cells. After 1 hour incubation at 37° C., the KRBH wasrecovered for measurement of glucose-induced insulin secretion and thecells were extracted with acid-ethanol (see also Irminger, J.-C. et al.,2003, Endocrinology 144: 1368-1379). Insulin levels were determined byan Enzyme-Linked Immunosorbent Assay (ELISA) for mouse insulin(Mercodia) and performed according to the manufacture isrecommendations. An alternative medium for proper insulin release wasmedium based on DMEM with glucose concentration of 1 g/l (Gibco)supplemented with non-essential amino acids (Gibco, stock solution1:100) and additional factors mentioned above. Such medium can beapplied 1 to 6 days before use of the cells.

A basal insulin secretion is expected when both wild type and neurturininduced insulin-producing cells are cultured in low glucoseconcentrations (2.8 mM). However, the neurturin inducedinsulin-producing cells highly respond to glucose stimulation. In thepresence of high glucose concentrations (16.7 mM), an increase ininsulin secretion is expected in neurturin ES derived insulin-producingcells.

Example 6 Transplantation of Pax4 ES Derived Insulin-Producing Cells inSTZ Diabetic Mice

The therapeutic potential of neurturin induced insulin-producing cellsto improve and cure diabetes can be investigated by transplanting thecells into streptozotocin induced diabetic mice. Streptozotocin is anantibiotic which is cytotoxic to beta-cells when administered at certaindosage (see Rodrigues et al.: Streptozotocin-induced diabetes, inMcNeill (ed) Experimental Models of Diabetes, CRC Press LLC, 1999). Itseffect is rapid, rendering an animal severely diabetic within 48 hours.

Non-fasted Male BalbC mice were treated with STZ to develophyperglycaemia after STZ treatment. Mice were considered diabetic ifthey had a blood glucose level above 10 mmol/l for more than 3consecutive days. Cells were transplanted under the kidney capsule andinto the spleen of animals. The presence of the insulin-producing cellswas confirmed by immunohistological analysis of the transplanted tissue.Results are expected to demonstrate that the transplanted cells cannormalise blood glucose in diabetic animals.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

Example 7 Generation of a mDG770 Transgenic Construct

A complete mDG770 Open Reading Frame (ORF) was cloned under the controlof the rat insulin promoter II (Lomedico at al., (1979) Cell 18:545-558) using the Gateway system (Invitrogen). For the structure of thetransgenic construct, see also FIG. 2.

Example 8 Generation of rIP-mDG770 Transgenic Mice

Transgenic construct DNA (see Example 7) was injected into C57/BL6×CBAembryos (Harlan Winkelmann, Borchen, Germany) using standard techniques(see, for example, Brinster at al. (1985), Proc. Natl. Acad. Sci. USA82: 4438-4442). The mDG770 transgene (see Example 7) was expressed underthe control of the rat insulin promoter II (Lomedico at al., supra)using techniques known to those skilled in the art (for example, see,Gunnig at al. (1987), Proc. Natl. Acad. Sci. USA 84, 4831-4835). Usingthis technique, several independent founderlines were generated.

Example 9 Genotype Analysis of rIP-mDG770 Transgenic Mice

Genotyping was performed by PCR using genomic DNA isolated from the tailtip. To detect the mDG770 transgene a transgene specific forward primer(5′ tgc taat ctg tot gga tgt gee 3° and a mDG770 transgene specificreverse primer (5′ aag gao acc tcg tcc tca tag 3′) was used.

Example 10 mDG770 Expression Analysis Via TaqMan Analysis

The expression of the mDG770 transgene in islets was monitored by TaqMananalysis. For this analysis, 25 ng cDNA derived from pancreatic isletRNA isolated from transgenic mice and their littermates and a mDG770specific primer/probe pair were used to detect endogenous as well astransgenic mDG770 expression (mDG770-1 forward primer: 5′ GCC TAT GAGGAC GAG GTG TCC 3′, mDG770 reverse primer: 5′ AGC TCT TGC AGC GTG TGG T3°, mDG770 probe: 5′ TCC TGG ACG TGC aca GCC GC 3′). TaqMan analysis wasperformed using standard techniques known to those skilled in the art.Ectopic transgene expression was detected in 3 of 4 rIP-mDG770transgenic founderlines analysed. The two founderlines showing highesttransgene expression levels were used for further analysis. For thelevel of mDG70 expression in islets of a transgenic animal compared to awild-type animal, see also FIG. 3.

Example 11 Analytical Procedures Performed in mDG770 Transgenic Mice

3 to 6 mice were housed per cage and were provided with food ad libitum.Metabolic blood parameters were determined using venous blood isolatedfrom tail vein or via retroorbital bleeding. Blood glucose values weredetermined using One Touch blood glucose meters (LifeScan, Germany),mDG770 transgenic mice exhibit reduced random fed blood glucose levels(rIP-mDG770), compared to wild type (wt) mice, see also FIG. 4.

1-63. (canceled)
 64. A method for promoting regeneration of pancreaticbeta-cells in a mammal having impaired beta-cell function, comprisingthe step of: administering a neurturin product to said mammal in anamount sufficient to increase neurturin concentration relative to anuntreated mammal having impaired beta-cell function, wherein the numberof functional beta-cells in said mammal administered the neurturinproduct are increased; wherein the neurturin product comprises abiologically active neurturin polypeptide; wherein the biologicallyactive neurturin polypeptide is capable of dimerizing with anotherneurturin polypeptide; and wherein the biologically active neurturinpolypeptide shares at least 90% sequence identity with: a) the humanneurturin precursor protein having the amino acid sequence set forth inSEQ ID NO: 7; or b) the mature neurturin protein product that resultsfrom the cleavage of the human neurturin protein precursor having anamino acid sequence set forth in SEQ ID NO:
 7. 65. A method forstimulating and/or inducing the differentiation of insulin-producingcells from progenitor cells comprising contacting said progenitor cellswith a neurturin product; wherein said neurturin product comprises abiologically active neurturin polypeptide; wherein said biologicallyactive neurturin polypeptide is capable of dimerizing with anotherneurturin polypeptide; and wherein said biologically active neurturinpolypeptide shares at least 90% sequence identity with: a) the humanneurturin precursor protein having the amino acid sequence set forth inSEQ ID NO: 7; or b) the mature neurturin protein product that resultsfrom the cleavage of the human neurturin protein precursor having anamino acid sequence set forth in SEQ ID NO:
 7. 66. The method of claim65, wherein said progenitor cells are in vitro or ex vivo.
 67. Themethod of claim 65, wherein said progenitor cells are in an animalhaving impaired beta-cell function, and wherein said contactingcomprises administering said neurturin product to said animal.
 68. Amethod for treating a mammal having impaired beta-cell function, whereinsaid method comprises the steps of: a) administering a neurturin productto said mammal in an amount sufficient to increase neurturinconcentration relative to an untreated mammal having impaired beta-cellfunction; wherein the neurturin product comprises a biologically activeneurturin polypeptide; wherein the biologically active neurturinpolypeptide is capable of dimerizing with another neurturin polypeptide;and wherein the biologically active neurturin polypeptide shares atleast 90% sequence identity with: i) the human neurturin precursorprotein having the amino acid sequence set forth in SEQ ID NO: 7; or ii)the mature neurturin protein product that results from the cleavage ofthe human neurturin protein precursor having an amino acid sequence setforth in SEQ ID NO: 7; b) administering to said mammal animmunosuppressive agent.
 69. The method of claim 65, wherein saidneurturin product and said immunosuppressive agent are coadministered.70. The method of claim 65, wherein said neurturin product and saidimmunosuppressive agent are administered to said mammal separately. 71.A method for normalizing blood glucose levels in a mammal havingimpaired beta-cell function, comprising the step of: administering aneurturin product to said mammal in an amount sufficient to increaseneurturin concentration relative to an untreated mammal having impairedbeta-cell function, wherein the levels of blood glucose are normalizedin said mammal administered the neurturin product; wherein the neurturinproduct comprises a biologically active neurturin polypeptide; whereinthe biologically active neurturin polypeptide is capable of dimerizingwith another neurturin polypeptide; and wherein the biologically activeneurturin polypeptide shares at least 90% sequence identity with: a) thehuman neurturin precursor protein having the amino acid sequence setforth in SEQ ID NO: 7; or b) the mature neurturin protein product thatresults from the cleavage of the human neurturin protein precursorhaving an amino acid sequence set forth in SEQ ID NO:
 7. 72. A methodfor generating insulin-producing cells from progenitor cells comprisingthe steps of: a) obtaining embryonic stem (ES) cells expressing a Pax4gene; and b) culturing said Pax4-expressing ES cells in the presence ofa neurturin product, wherein said neurturin product comprises abiologically active neurturin polypeptide; wherein said biologicallyactive neurturin polypeptide is capable of dimerizing with anotherneurturin polypeptide; and wherein said biologically active neurturinpolypeptide shares at least 90% sequence identity with: i) the humanneurturin precursor protein having the amino acid sequence set forth inSEQ ID NO: 7; or ii) the mature neurturin protein product that resultsfrom the cleavage of the human neurturin protein precursor having anamino acid sequence set forth in SEQ ID NO: 7; wherein culturing saidPax-4 expressing ES cells in the presence of said neurturin productstimulates and/or induces differentiation of insulin-producing cells.73. A method for generating insulin-producing cells from progenitorcells, comprising the steps of: a) contacting cells that express apancreatic gene with a neurturin product; wherein the neurturin productcomprises a biologically active neurturin polypeptide; wherein thebiologically active neurturin polypeptide is capable of dimerizing withanother neurturin polypeptide; and wherein the biologically activeneurturin polypeptide shares at least 90% sequence identity with: i) thehuman neurturin precursor protein having the amino acid sequence setforth in SEQ ID NO: 7; or ii) the mature neurturin protein product thatresults from the cleavage of the human neurturin protein precursorhaving an amino acid sequence set forth in SEQ ID NO: 7; and b)culturing said pancreatic gene-expressing cells in the presence of saidneurturin product; wherein culturing said pancreatic gene-expressingcells in the presence of said neurturin product stimulates and/orinduces differentiation of insulin-producing cells.
 74. The method ofclaim 64, wherein the biologically active neurturin polypeptide sharesat least 95% sequence identity with: a) the human neurturin precursorprotein having the amino acid sequence set forth in SEQ ID NO: 7; or b)the mature neurturin protein product that results from the cleavage ofthe human neurturin protein precursor having an amino acid sequence setforth in SEQ ID NO:
 7. 75. The method of claim 68, wherein thebiologically active neurturin polypeptide shares at least 95% sequenceidentity with: a) the human neurturin precursor protein having the aminoacid sequence set forth in SEQ ID NO: 7; or b) the mature neurturinprotein product that results from the cleavage of the human neurturinprotein precursor having an amino acid sequence set forth in SEQ ID NO:7.
 76. The method of claim 64, wherein said mammal having impairedbeta-cell function suffers from diabetes type I.
 77. The method of claim68, wherein said mammal having impaired beta-cell function suffers fromdiabetes type I.
 78. The method of claim 64, wherein said mammal havingimpaired beta-cell function suffers from diabetes type II.
 79. Themethod of claim 68, wherein said mammal having impaired beta-cellfunction suffers from diabetes type II.
 80. The method of claim 64,wherein said mammal having impaired beta-cell function suffers fromlatent autoimmune diabetes in adults.
 81. The method of claim 68,wherein said mammal having impaired beta-cell function suffers fromlatent autoimmune diabetes in adults.
 82. The method of claim 64,wherein the biologically active neurturin polypeptide shares 100%sequence identity with: a) the human neurturin precursor protein havingthe amino acid sequence set forth in SEQ ID NO: 7; or b) the matureneurturin protein product that results from the cleavage of the humanneurturin protein precursor having an amino acid sequence set forth inSEQ ID NO:
 7. 83. The method of claim 68, wherein the biologicallyactive neurturin polypeptide shares 100% sequence identity with: a) thehuman neurturin precursor protein having the amino acid sequence setforth in SEQ ID NO: 7; or b) the mature neurturin protein product thatresults from the cleavage of the human neurturin protein precursorhaving an amino acid sequence set forth in SEQ ID NO: 7.